Derivatives of A-30912A nucleus

ABSTRACT

Compounds of the formula ##STR1## wherein R 1  is an N-alkanoyl amino acyl group of the formula ##STR2## wherein: W is a divalent aminoacyl radical of the formula: ##STR3## wherein A is C 1  -C 10  alkylene or C 5  -C 6  cycloalkylene; ##STR4## wherein R 3  is hydroxymethyl, hydroxyethyl, mercaptomethyl, mercaptoethyl, methylthioethyl, 2-thienyl, 3-indole-methyl, phenyl, benzyl, or substituted phenyl or substituted benzyl in which the benzene ring thereof is substituted with chloro, bromo, iodo, nitro, C 1  -C 3  alkyl, hydroxy, C 1  -C 3  alkylthio, carbamyl, or C 1  -C 3  alkylcarbamyl; ##STR5## wherein X is hydrogen, chloro, bromo, iodo, nitro, C 1  -C 3  alkyl, hydroxy, C 1  -C 3  alkoxy, mercapto, C 1  -C 3  alkylthio, carbamyl, or C 1  -C 3  alkylcarbamyl; ##STR6## wherein X 1  is chloro, bromo, or iodo; ##STR7## wherein B is a divalent radical of the formula: --(CH 2 ) n  --, wherein n is an integer from 1 to 3; --CH═CH--; --CH═CH--CH 2  --; ##STR8## and R 2  is C 1  -C 17  alkyl or C 2  -C 17  alkenyl; have antifungal activity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.103,149, filed Dec. 13, 1979, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to novel semisynthetic antifungal compounds whichare prepared by the acylation of the cyclic peptide nucleus produced bythe enzymatic deacylation of antibiotic A30912 factor A.

Antibiotic A-30912 factor A is an antifungal cyclic peptide having theformula: ##STR9## wherein R is the linoleoyl group ##STR10## Throughoutthis application, the cyclic peptide formulas, such as formula I, assumethat the amino acids represented are in the L-configuration. The factoris isolated from the A30912 complex which contains other factorsarbitrarily designated factors B, C, D, E, and G. The A-30912 complexand the individual factors A through G are described by M. Hoehn and K.Michel in U.S. Pat. No. 4,024,245. Factor A is identical to antibioticA-22802 which is described by C. Higgins and K. Michel in U.S. Pat. No.4,024,246. Factor A has also been found to be identical to antibioticechinocandin B [see F. Benz et al., Helv. Chim. Acta, 57, 2459 (1974)and Swiss Pat. No. 568,386] and to antibiotic SL 7810/F [see C.Keller-Juslen et al. Tetrahedron Letters, 4147 (1976) and Belgium Pat.No. 834,289].

Antibiotic A-30912 factor A is prepared by fermentation using one ofseveral different organisms, namely: (a) Aspergillus rugulosus NRRL 8113(see U.S. Pat. No. 4,024,245); (b) Aspergillus nidulans NRRL 8112 (seeU.S. Pat. No. 4,024,246); (c) Aspergillus nidulans var. echinulatusA-32204 as described in Swiss Pat. No. 568,386; (d) Aspergillusrugulosus NRRL 8039 (see Belgian Pat. No. 834,289); or (e) Aspergillusnidulans var. roseus NRRL 11440 (see co-pending application of L. Boeckand R. Kastner, METHOD OF PRODUCING THE A-30912 ANTIBIOTICS, Ser. No.126,078, filed Mar. 3, 1980, which is a continuation-in-part ofapplication Ser. No. 46,744, filed June 8, 1979, (now abandoned), theentire disclosure of which is incorporated herein by reference.)

A subculture of A. nidulans var. roseus has been deposited and made apart of the permanent culture collection of the Northern RegionalResearch Laboratory, U.S. Department of Agriculture, AgriculturalResearch Service, Peoria, Ill. 61604, from which it is available to thepublic under the number NRRL 11440.

When a strain of A. nidulans var. roseus NRRL 11440 is used to produceA-30912 factor A, a complex of factors is obtained which for convenienceis called the A-42355 antibiotic complex. A-30912 factor A is the majorfactor of the A-42355 antibiotic complex, while factors B, D and H areminor factors. Examples 96, 97, and 98, herein, illustrate thepreparation of the A-42355 complex and the isolation and purification ofA-30912 factor A therefrom. A-30912 factor H is further described in aco-pending application of Karl. H. Michel entitled ANTIBIOTIC A-30912FACTOR H, Ser. No. 117,739, filed Feb. 1, 1980, which is acontinuation-in-part of application Ser. No. 46,875, filed June 8, 1979(now abandoned).

In the A-30912 factor A molecule (Formula I), the linoleoyl side chain(R) is attached at the cyclic peptide nucleus at the α-amino group ofthe dihydroxyornithine residue. Surprisingly, it has been found that thelinoleoyl side chain can be cleaved from the nucleus by an enzymewithout affecting the chemical intregity of the nucleus. The enzymeemployed to effect the deacylation reaction is produced by amicroorganism of the family Actinoplanaceae, preferably themicroorganism Actinoplanes utahensis NRRL 12052, or a variant thereof.To accomplish deacylation, antibiotic A30912 factor A is added to aculture of the microorganism and the culture is allowed to incubate withthe substrate until the deacylation is substantially complete. Thecyclic nucleus thereby obtained is separated from the fermentation brothby methods known in the art. Unlike antibiotic A-30912 factor A, thecyclic nucleus (lacking the linoleoyl side chain) is substantiallydevoid of antifungal activity.

The cyclic nucleus afforded by the aforedescribed enzymatic deacylationof antibiotic A-30912 factor A, is depicted in Formula II. ##STR11##

Removal of the side chain group affords a free primary α-amino group inthe dihydroxyornithine residue of the cyclic peptide. For convenience,the compound having the structure given in Formula II will be referredto herein as "A-30912A nucleus." As will be apparent to those skilled inthe art, A-30912A nucleus can be obtained either in the form of the freeamine or of the acid addition salt. Although any suitable acid additionsalt may be employed, those which are non-toxic and pharmaceuticallyacceptable are preferred.

The method of preparing A-30912A nucleus from antibiotic A-30912 factorA by means of fermentation using Actinoplanes utahensis NRRL 12052 isdescribed in the co-pending application of Bernard J. Abbott and DavidS. Fukuda, entitled "A-30912A NUCLEUS", Ser. No. 103,017, which wasfiled Dec. 13, 1979. A continuation-in-part application of thisapplication, with the corresponding Ser. No. 103,149, is being filedherewith this even date, the full disclosure of which is incorporatedherein by reference. Example 93, herein, illustrates the preparation ofA-30912A nucleus by fermentation using antibiotic A-30912 factor A asthe substrate and Actinoplanes utahensis NRRL 12052 as themicroorganism.

The enzyme produced by Actinoplanes utahensis NRRL 12052 may be the sameenzyme which has been used to deacylate penicillins (see Walter J.Kleinschmidt, Walter E. Wright, Frederick W. Kavanagh, and William M.Stark, U.S. Pat. No. 3,150,059, issued Sept. 22, 1964).

Cultures of representative species of Actinoplanaceae are available tothe public from the Northern Regional Research Laboratory under thefollowing accession numbers:

Actinoplanes utahensis: NRRL 12052

Actinoplanes missouriensis: NRRL 12053

Actinoplanes sp.: NRRL 8122

Actinoplanes sp.: NRRL 12065

Streptosporangium roseum var. hollandensis: NRRL 12064

The effectiveness of any given strain of microorganism within the familyActinoplanaceae for carrying out the deacylation of this invention isdetermined by the following procedure. A suitable growth medium isinoculated with the microorganism. The culture is incubated at about 28°C. for two or three days on a rotary shaker. One of the substrateantibiotics is then added to the culture. The pH of the fermentationmedium is maintained at about pH 6.5. The culture is monitored foractivity using a Candida albicans assay. Loss of antibiotic activity isan indication that the microorganism produces the requisite enzyme fordeacylation. This must be verified, however, using one of the followingmethods: (1) analysis by HPLC for presence of the intact nucleus; or (2)reacylation with an appropriate side chain (e.g. linoleoyl, stearoyl, orpalmitoyl) to restore activity.

It is known that other antibiotic substances possess the same nucleus asthat of antibiotic A-30912 factor A. These antibiotics differ fromantibiotic A-30912 factor A in that different acyl groups are present inplace of the linoleoyl group (R) in Formula I. Such antibiotics are: (a)tetrahydro-A-30912 factor A (tetrahydro-SL 7810/F; tetrahydroechinocandin B) described in Belgium Pat. No. 834,289 and by F. Benz etal., Helv. Chim. Acta, 57 2459 (1974), which compound is depicted inFormula I when R is stearoyl; and (b) aculaecin A, which is a componentof the aculaecin complex (prepared by fermentation using Aspergillusaculeatus NRRL 8075) and is described by K. Mizuno et al., in U.S. Pat.No. 3,978,210. As is discussed in Belgium Pat. No. 859,067, in aculaecinA the palmitoyl side chain is present in place of linoleoyl.Tetrahydro-A-30912 factor A can be prepared from antibiotic A-30912factor A by catalytic hydrogenation using PtO₂ in ethanol under positivepressure. Both tetrahydro-A-30912 factor A and aculaecin A can beemployed as substrates for the enzymatic deacylation using theprocedures herein described.

SUMMARY OF THE INVENTION

The invention sought to be patented comprehends novel compounds derivedby acylating the A-30912A nucleus (Formula II). The compounds of thepresent invention have the chemical structure depicted in Formula III:##STR12## wherein R¹ is an N-alkanoyl amino acyl group of the formula##STR13## wherein: W is a divalent aminoacyl radical of the formula:##STR14## wherein A is C₁ -C₁₀ alkylene or C₅ -C₆ cycloalkylene;##STR15## wherein R³ is hydroxymethyl, hydroxyethyl, mercaptomethyl,mercaptoethyl, methylthioethyl, 2-thienyl, 3-indole-methyl, phenyl,benzyl, or substituted phenyl or substituted benzyl in which the benzenering thereof is substituted with chloro, bromo, iodo, nitro, C₁ -C₃alkyl, hydroxy, C₁ -C₃ alkylthio, carbamyl, or C₁ -C₃ alkylcarbamyl;##STR16## wherein X is hydrogen chloro, bromo, iodo, nitro, C₁ -C₃alkyl, hydroxy, C₁ -C₃ alkoxy, mercapto, C₁ -C₃ alkylthio, carbamyl, orC₁ -C₃ alkylcarbamyl; ##STR17## wherein X¹ is chloro, bromo, or iodo;##STR18## wherein B is a divalent radical of the formula: --(CH₂)_(n)--, wherein n is an integer from 1 to 3; --CH═CH--; --CH═CH--CH₂ --; or##STR19## and R² is C₁ -C₁₇ alkyl or C₂ -C₁₇ alkenyl.

As employed herein the terms "alkylene", "alkyl", "alkoxy", "alkylthio",and "alkenyl" comprehend both straight and branched hydrocarbon chains."Alkyl" means a univalent saturated hydrocarbon radical. "Alkenyl" meansa univalent unsaturated hydrocarbon radical containing one, two, orthree double bonds, which may be oriented in the cis or transconfiguration. "Alkylene" means a divalent saturated hydrocarbonradical. "Cycloalkylene" means a divalent cyclic saturated hydrocarbonradical.

Illustrative C₁ -C₁₀ alkylene radicals, which are preferred for purposesof this invention are:

--CH₂ --; ##STR20## in which R⁵ is C₁ -C₄ alkyl (i.e., methyl, ethyl,n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, or 1-methylpropyl);--(CH₂)_(m) in which m is an integer from 2 to 10; and ##STR21## inwhich p is an integer from 1 to 8 and q is an integer from 0 to 7,provided that n+m must be no greater than 8.

Illustrative C₁ -C₁₇ alkyl groups which are preferred for the purposesof this invention are:

(a) CH₃ --;

(b) --(CH₂)_(n) CH₃ wherein n is an integer from 1 to 16; and ##STR22##wherein r and s are independently, an integer from 0 to 14 provided thatr+s can be no greater than 14. Illustrative C₂ -C₁₇ alkenyl radicals,which are preferred for the purpose of this invention, are

(a) --(CH₂)_(t) --CH═CH--(CH₂)_(u) --CH₃ wherein t and u areindependently, an integer from 0 to 14 provided that t+u can be nogreater than 14.

(b) --(CH₂)_(v) --CH═CH--(CH₂)_(y) --CH═CH--(CH₂)_(z) --CH₃ wherein vand z are independently, an integer from 0 to 11 and y is an integerfrom 1 to 12 provided that v+y+z can be no greater than 11.

In particular, the following embodiments of the C₁ -C₁₇ alkyl groups arepreferred:

CH₃ --

CH₃ (CH₂)₅ --

CH₃ (CH₂)₆ --

CH₃ (CH₂)₈ --

CH₃ (CH₂)₁₀ --

CH₃ (CH₂)₁₂ --

CH₃ (CH₂)₁₄ --

CH₃ (CH₂)₁₆ --

In particular, the following embodiments of the C₂ -C₁₇ alkenyl groupsare preferred:

cis-CH₃ (CH₂)₅ CH═CH(CH₂)₇ --

trans-CH₃ (CH₂)₅ CH═CH(CH₂)₇ --

cis-CH₃ (CH₂)₁₀ CH═CH(CH₂)₄ --

trans-CH₃ (CH₂)₁₀ CH═CH(CH₂)₄ --

cis-CH₃ (CH₂)₇ CH═CH(CH₂)₇ --

trans-CH₃ (CH₂)₇ CH═CH(CH₂)₇ --

cis-CH₃ (CH₂)₅ CH═CH(CH₂)₉ --

trans-CH₃ (CH₂)₅ CH═CH(CH₂)₉ --

cis, cis-CH₃ (CH₂)₄ CH═CHCH₂ CH═CH(CH₂)₇ --

trans, trans-CH₃ (CH₂)₄ CH═CHCH₂ CH═CH(CH₂)₇ --

cis,cis,cis-CH₃ CH₂ CH═CHCH₂ CH═CHCH₂ CH═CH--(CH₂)₇ --.

When "W" is a divalent radical of the formula ##STR23## it will berecognized by those skilled in the art that the ##STR24## function andthe --NH-- function may be oriented on the benzene ring in the ortho,meta, or para configuration relative to each other. The substituentrepresented by X may be substituted at any available position of thebenzene ring. Preferred embodiments are those in which X is hydrogen andthe ##STR25## and --NH-- functions are oriented in the paraconfiguration.

The terms "substituted phenyl" and "substituted benzyl", as defined byR₃ in Formula III, contemplate substitution of a group at any of theavailable positions in the benzene ring--i.e. the substituent may be inthe ortho, meta, or para configuration. The term "C₁ -C₃ alkyl" asdefined by R₃ or X in Formula III includes the methyl, ethyl, n-propyl,or i-propyl groups.

DETAILED DESCRIPTION OF THE INVENTION

The compounds of Formula III inhibit the growth of pathogenic fungi asevidenced by standard biological test procedures. The compounds areuseful, therefore, for controlling the growth of fungi on environmentalsurfaces (as an antiseptic) or in treating infections caused by fungi.The antifungal activity of the compounds has been demonstrated againstCandida albicans in vitro in agar plate disc diffusion tests and in agardilution tests, or in vivo in tests in mice infected with C. albicans.Thus, the compounds are particularly useful in treating infectionscaused by strains of C. albicans (candidosis). The compounds of FormulaIII have also shown activity in vitro in agar-plate disc diffusion testsagainst Trichophyton mentagrophytes, a dermatophytic organism. Activityhas also been found in in vitro agar plate disc diffusion tests againstSaccharomyces pastorianus, and Neurospora crassa. Certain compounds (asshown in Example 92, Table 8) give significant blood levels upon oraladministration in mice.

When given to a dog by intravenous administration, 100 mg/kg per day forfive days, the compound of Formula III wherein R isn-dodecanoyl-p-aminobenzoyl showed no outward signs of toxicity,although increased SGPT levels were observed.

The compounds of Formula III are prepared by acylating A-30912A nucleusat the α-amino group of dihydroxyornithine with the appropriateN-alkanoyl aminoacyl or N-alkenoyl amino acyl side chain using methodsconventional in the art for forming an amide bond. The acylation isaccomplished, in general, by reacting the nucleus with an activatedderivative of the acid (Formula IV) corresponding to the desired acylside chain group. ##STR26## (W and R² have the meaning described hereinsupra). By the term "activated derivative" is meant a derivative whichrenders the carboxyl function of the acylating agent reactive tocoupling with the primary amino group to form the amide bond which linksthe acyl side chain to the nucleus. Suitable activated derivatives,their methods of preparation, and their methods of use as acylatingagents for a primary amine will be recognized by those skilled in theart. Preferred activated derivatives are: (a) an acid halide (e.g. acidchloride), (b) an acid anhydride (e.g. an alkoxyformic acid anhydride oraryloxyformic acid anhydride) or (c) an activated ester (e.g. a2,4,5-trichlorophenyl ester, a N-hydroxybenztriazole ester, or anN-hydroxysuccinimide ester). Other methods for activating the carboxylfunction include reaction of the carboxylic acid with a carbonyldiimide(e.g. N,N-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide) togive a reactive intermediate which, because of instability, is notisolated, the reaction with the primary amine being carried out in situ.

A preferred method for preparing the compounds of Formula III is by theactive ester method. The use of the 2,4,5-trichlorophenyl ester of thedesired N-alkanoylamino acid or N-alkenoylamino acid (Formula IV) as theacylating agent is most preferred. In this method, an excess amount ofthe active ester is reacted with the nucleus at room temperature in anonreactive organic solvent such as dimethyl formamide (DMF). Thereaction time is not critical, although a time of about 15 to about 18hours is preferred. At the conclusion of the reaction, the solvent isremoved, and the residue is purified such as by column chromatographyusing silica gel as the stationary phase and a mixture of ethylacetate/methanol (3:2, v/v) as the solvent system.

The 2,4,5-trichlorophenyl esters of the N-alkanoylamino acids orN-alkenoylamino acids can be prepared conveniently by treating thedesired amino acid (Formula IV) with 2,4,5-trichlorophenol in thepresence of a coupling agent, such as N,N'-dicyclohexylcarbodiimide.Other methods suitable for preparing amino acid esters will be apparentto those skilled in the art.

The N-alkanoylamino acids or N-alkenoylamino acids are either knowncompounds or they can be made by acylating the appropriate amino acidwith the appropriate alkanoyl or alkenoyl group using conventionalmethods, such as those described herein supra. A preferred way ofpreparing the N-alkanoylamino acids is by treating the appropriate aminoacid with an alkanoic acid chloride in pyridine. The alkanoic acids oralkenoic acids, the activated derivatives thereof, and the amino acidsemployed in the preparation of the products of this invention are eitherknown compounds or they can be made by known methods or by modificationof known methods which will be apparent to those skilled in the art.

If a particular amino acid contains an acylable functional group otherthan the amino group, it will be understood by those skilled in the artthat such a group must be protected prior to reaction of the amino acidwith the reagent employed to attach the alkanoyl or alkenoyl group.Suitable protecting groups can be any group known in the art to beuseful for the protection of a side chain functional group in peptidesynthesis. Such groups are well known, and the selection of a particularprotecting group and its method of use will be readily known to oneskilled in the art [see, for example, "Protective Groups In OrganicChemistry", M. McOmie, Editor, Plenum Press, N.Y., 1973].

It will be recognized that certain amino acids employed in the synthesisof the products of this invention may exist in optically active forms,and both the natural configuration (L-configuration) and unnaturalconfiguration (D-configuration) may be employed as starting materialsand will give products which are within the contemplation of thisinvention.

When employed systemically, the dosage of the compounds of Formula IIIwill vary according to the particular compound being employed, theseverity and nature of the infection, and the physical condition of thesubject being treated. Therapy should be initiated at low dosages, thedosage being increased until the desired antifungal effect is obtained.The compounds can be administered intravenously or intramuscularly byinjection in the form of a sterile aqueous solution or suspension towhich may be added, if desired, various conventional pharmaceuticallyacceptable preserving, buffering, solubilizing, or suspending agents.Other additives, such as saline or glucose may be added to make thesolutions isotonic. The proportions and nature of such additives will beapparent to those skilled in the art.

Certain compounds of Formula III give significant blood levels afteroral administration (see Example 92, Table 8) and can be administeredsystemically by the oral route. For oral use, such compounds can beadministered in combination with pharmaceutically acceptable carriers orexcipients in the form of capsules, tablets or powders. The nature andproportion of such carriers or excipients of which will be recognized bythose skilled in the art.

When employed to treat vaginal candida infections, the compounds ofFormula III can be administered in combination with pharmaceuticallyacceptable conventional excipients suitable for intravaginal use.Formulations adapted for intravaginal administration will be known tothose skilled in the art.

The methods of making and using the compounds of the present inventionare illustrated in the following examples

EXAMPLES 1-30

Table 1, below, gives the preparation of various N-alkanoyl amino acids.The compounds shown in Table 1 are prepared according to the followinggeneral procedure:

The appropriate alkanoic acid chloride is added dropwise to theappropriate amino acid (1:1 mole ratio) dissolved in pyridine. Theamount of pyridine employed should be such as to make the concentrationof reactants between 0.1 to 0.2 M. The solution is stirred at roomtemperature for about 3 to 6 hours, after which it is poured into alarge volume of water. The product precipitates from solution and iscollected by filtration and crystallized from methanol.

                                      TABLE 1                                     __________________________________________________________________________    Preparation of N-alkanoyl Amino Acids                                         __________________________________________________________________________    Example                                                                            Alkanoic acid chloride                                                                       Amino Acid                                                No.  Formula  wt.   Formula           wt.                                     __________________________________________________________________________    1    CH.sub.3 (CH.sub.2).sub.10 COCl                                                        3.00                                                                             g  NH.sub.2 CH(CH.sub.2 C.sub.6 H.sub.5)CO.sub.2                                                   2.0                                                                              g.                                   2    CH.sub.3 (CH.sub.2).sub.10 COCl                                                        234                                                                              mg NH.sub.2 (CH).sub.4 CO.sub.2 H                                                                  482                                                                              mg.                                  3    CH.sub.3 (CH.sub.2).sub.10 COCl                                                        21.85                                                                            g. NH.sub.2 (CH.sub.2).sub.10 CO.sub.2 H                                                           20.1                                                                             mg.                                  4    CH.sub.3 (CH.sub.2).sub.10 COCl                                                        11.09                                                                            g.                                                                                ##STR27##        6.2                                                                              g.                                   5    CH.sub.3 (CH.sub.2).sub.10 COCl                                                        2.19                                                                             g.                                                                                ##STR28##        1.37                                                                             g.                                   6    CH.sub.3 (CH.sub.2).sub.10 COCl                                                        437                                                                              mg.                                                                               ##STR29##        306                                                                              mg.                                  7    CH.sub.3 (CH.sub.2).sub.10 COCl                                                        2.17                                                                             g.                                                                                ##STR30##        2.06                                                                             g.                                   8    CH.sub.3 (CH.sub.2).sub.10 COCl                                                        2.17                                                                             g.                                                                                ##STR31##        3.89                                                                             g.                                   9    CH.sub.3 (CH.sub.2).sub.10 COCl                                                        2.17                                                                             g.                                                                                ##STR32##        1.51                                                                             g.                                   10   CH.sub.3 (CH.sub.2).sub.10 COCl                                                        2.17                                                                             g.                                                                                ##STR33##        1.51                                                                             g.                                   11   CH.sub.3 (CH.sub.2).sub.10 COCl                                                        2.17                                                                             g.                                                                                ##STR34##        1.67                                                                             g.                                   12   CH.sub.3 (CH.sub.2).sub.10 COCl                                                        21.85                                                                            g.                                                                                ##STR35##        15.1                                                                             g.                                   13   CH.sub.3 (CH.sub.2).sub.10 COCl                                                        2.19                                                                             g.                                                                                ##STR36##        1.6                                                                              g.                                   14   CH.sub.3 (CH.sub.2).sub.10 COCl                                                        21.85                                                                            g.                                                                                ##STR37##        19.4                                                                             g.                                   15   CH.sub.3 (CH.sub.2).sub.10 COCl                                                        8.52                                                                             g.                                                                                ##STR38##        5.4                                                                              g.                                   16   CH.sub.3 (CH.sub.2).sub.5 COCl                                                         14.85                                                                            g. NH.sub.2 (CH.sub.2).sub.10 CO.sub.2 H                                                           20.1                                                                             g.                                   17   CH.sub.3 COCl                                                                          785                                                                              mg.                                                                               ##STR39##        1.37                                                                             g.                                   18   CH.sub.3 (CH.sub.2).sub.5 COCl                                                         1.49                                                                             g.                                                                                ##STR40##        1.37                                                                             g.                                   19   CH.sub.3 (CH.sub.2).sub.8 COCl                                                         1.91                                                                             g.                                                                                ##STR41##        1.37                                                                             g.                                   20   CH.sub.3 (CH.sub.2).sub.12 COCl                                                        2.46                                                                             g.                                                                                ##STR42##        1.37                                                                             g.                                   21   CH.sub.3 (CH.sub.2).sub.14 COCl                                                        2.74                                                                             g.                                                                                ##STR43##        1.37                                                                             g.                                   22   CH.sub.3 (CH.sub.2).sub.6 COCl                                                         3.42                                                                             g.                                                                                ##STR44##        3.43                                                                             g.                                   23   CH.sub.3 (CH.sub.2).sub.10 COCl                                                        4.60                                                                             g.                                                                                ##STR45##        3.43                                                                             g.                                   24   CH.sub.3 (CH.sub.2).sub.6 COCl                                                         3.42                                                                             g.                                                                                ##STR46##        3.43                                                                             g.                                   25   CH.sub.3 (CH.sub.2).sub.10 COCl                                                        4.60                                                                             g.                                                                                ##STR47##        3.43                                                                             g.                                   26   CH.sub.3 (CH.sub.2).sub.6 COCl                                                         3.42                                                                             g.                                                                                ##STR48##        3.43                                                                             g.                                   27   CH.sub.3 (CH.sub.2).sub.10 COCl                                                        4.60                                                                             g.                                                                                ##STR49##        3.43                                                                             g.                                   28   CH.sub.3 (CH.sub.2).sub.6 COCl                                                         3.42                                                                             g.                                                                                ##STR50##        6.18                                                                             g.                                   29   CH.sub.3 (CH.sub.2).sub.8 COCl                                                         4.01                                                                             g.                                                                                ##STR51##        4.12                                                                             g.                                   30   CH.sub.3 (CH.sub.2).sub.12 COCl                                                        5.18                                                                             g.                                                                                ##STR52##        4.12                                                                             g.                                   __________________________________________________________________________             Example                                                                            N-Alkanoyl Amino Acid                                                    No.  Formula                 wt.                                     __________________________________________________________________________             1    CH.sub.3 (CH.sub.2).sub.10 CONHCH(C.sub.5 H.sub.5)CO.sub.2                    H                       2.5                                                                              g.                                            2    CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4 CO.sub.2                                              598                                                                              mg.                                           3    CH.sub. 3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10 CO.sub.2                    H                       19.97                                                                            g.                                            4                                                                                   ##STR53##              6.0                                                                              g.                                            5                                                                                   ##STR54##              3.19                                                                             g.                                            6                                                                                   ##STR55##              670                                                                              mg.                                           7                                                                                   ##STR56##              3.58                                                                             g.                                            8                                                                                   ##STR57##              5.23                                                                             g.                                            9                                                                                   ##STR58##              3.04                                                                             g.                                            10                                                                                  ##STR59##              2.87                                             11                                                                                  ##STR60##              3.34                                                                             g.                                            12                                                                                  ##STR61##              33.3                                                                             g.                                            13                                                                                  ##STR62##              2.76                                                                             g.                                            14                                                                                  ##STR63##              37.6                                                                             g.                                            15                                                                                  ##STR64##              7.6                                                                              g.                                            16   CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10 CO.sub.2                                              31.3                                                                             g.                                            17                                                                                  ##STR65##              1.56                                                                             g.                                            18                                                                                  ##STR66##              2.25                                                                             g.                                            19                                                                                  ##STR67##              2.52                                                                             g.                                            20                                                                                  ##STR68##              2.84                                                                             g.                                            21                                                                                  ##STR69##              3.16                                                                             g.                                            22                                                                                  ##STR70##              3.20                                                                             g.                                            23                                                                                  ##STR71##              2.89                                                                             g.                                            24                                                                                  ##STR72##              3.19                                                                             g.                                            25                                                                                  ##STR73##              4.26                                                                             g.                                            26                                                                                  ##STR74##              4.76                                                                             g.                                            27                                                                                  ##STR75##              6.23                                                                             g.                                            28                                                                                  ##STR76##              4.26                                                                             g.                                            29                                                                                  ##STR77##              3.638                                                                            g.                                            30                                                                                  ##STR78##              4.187                                                                            g.                                   __________________________________________________________________________

EXAMPLES 31-60

Table 2, below, gives the preparation of the 2,4,5-trichlorophenylesters of the N-alkanoyl amino acids shown in Table 1. The compounds setforth in Table 2 are prepared according to the following generalprocedure:

The N-alkanoylamino acid (1 mole), 2,4,5-trichlorophenol (1.1 mole), anddicyclohexylcarbodiimide (1 mole) are dissolved in methylene chloride,ether or tetrahydrofuran. The solution is stirred at room temperaturefor about 16 to about 20 hours after which it is filtered. The filtrateis taken to dryness, and the product is crystallized from eitheracetonitrilewater or diethyl ether-petroleum ether.

                                      TABLE 2                                     __________________________________________________________________________    Preparation of 2,4,5-trichlorophenyl esters                                          N-Alkanoyl Amino Acid        Wt. of 2,4,5-trichlorophenol              Example No.                                                                          Formula                 wt   ester product                             __________________________________________________________________________    31     CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO.sub.2            H                      333 mg.                                                                            500 mg.                                   32     CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4 CO.sub.2 H                                            598 mg.                                                                            955 mg                                    33     CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10 CO.sub.2 H                                           3.83 g.                                                                            1.02 g.                                   34                                                                                    ##STR79##              638 mg.                                                                            410 mg.                                   35                                                                                    ##STR80##              3.19 g.                                                                            2.43 g.                                   36                                                                                    ##STR81##              670 mg.                                                                            1.03 g.                                   37                                                                                    ##STR82##              3.58 g.                                                                            2.20 g.                                   38                                                                                    ##STR83##              5.23 g.                                                                            1.41 g.                                   39                                                                                    ##STR84##              3.04 g.                                                                            4.7 g.                                    40                                                                                    ##STR85##              2.87 g.                                                                            4.45 g.                                   41                                                                                    ##STR86##              3.34 g.                                                                            3.86 g.                                   42                                                                                    ##STR87##              3.33 g.                                                                            2.6 g.                                    43                                                                                    ##STR88##              2.76 g.                                                                            2.14 g.                                   44                                                                                    ##STR89##              3.76 g.                                                                            1.0 g.                                    45                                                                                    ##STR90##              3.28 g.                                                                            4.4 g.                                    46     CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10 CO.sub.2 H                                            4.8 g.                                                                             1.68 g.                                   47                                                                                    ##STR91##              895 mg.                                                                            1.48 g.                                   48                                                                                    ##STR92##              1.245 g.                                                                           1.59 g.                                   49                                                                                    ##STR93##              2.52 g.                                                                            2.97 g.                                   50                                                                                    ##STR94##              2.84 g.                                                                            2.44 g.                                   51                                                                                    ##STR95##              3.16 g.                                                                            1.33 g.                                   52                                                                                    ##STR96##              2.08 g.                                                                            2.436 g. (700 mg. after recryst.)         53                                                                                    ##STR97##              2.65 g.                                                                            2.373 g.                                  54                                                                                    ##STR98##              2.68 g.                                                                            1.619 g.                                  55                                                                                    ##STR99##              3.19 g.                                                                            1.605 g.                                  56                                                                                    ##STR100##             2.38 g.                                                                            1.716 g.                                  57                                                                                    ##STR101##             2.83 g.                                                                            1.575 g.                                  58                                                                                    ##STR102##             4.19 g.                                                                            2.02 g.                                   59                                                                                    ##STR103##             2.88 g.                                                                            3.507 g.                                  60                                                                                    ##STR104##             3.33 g.                                                                            1.897 g.                                  __________________________________________________________________________

EXAMPLES 61-90

Table 3, below, gives the preparation of the derivatives of A-30912Anucleus prepared from the N-alkanoyl amino acid 2,4,5-trichlorophenylesters set forth in Table 2. The compounds set forth in Table 3 areprepared in general according to the following procedure:

To A-30912A nucleus, dissolved in dimethylformamide (DMF) is added the2,4,5-trichlorophenyl ester of the N-alkanoyl amino acid. The reactionmixture is stirred for 15-18 hours after which it is taken to dryness togive a residue. The residue is washed (two times each) with ethyl etherand by methylene chloride. The washings are discarded. The remainingresidue is dissolved in ethyl acetate-methanol (3:2, v/v) and ischromatographed on a silica gel (Woelm 70-150 mesh) column using theaforesaid solvent system as the eluent. The fractions from thechromatograph are monitored by TLC on silica gel (Merck) using ethylacetate-methanol (3:2, v/v) as the solvent system. Fractions containingthe desired product are combined, and solvent is removed to give theproduct as a residue. The product may be analyzed by reversed phase HPLCas follows: The sample dissolved in H₂ O/CH₃ OH/CH₃ CN (1:2:2 v/v)(1mg./ml.) is injected into a 1/4 inch by 12 inch stainless steel columnpacked with C₁₈ Micro Bondapak resin (Waters Associates, Inc., Milford,Mass) and the column is eluted with a solvent system comprising H₂ O/CH₃OH/CH₃ CN (1:2:2 v/v). The elution is ml./minute using a Waters 600Apump (Waters Associates, Inc.) and chart speed of 0.2 in./minute. Eluentis monitored with a Varian Vari-Chrom UV detector at 230 nm.

The products may also be analyzed by field desorption mass spectrometry(FDMS).

                                      TABLE 3                                     __________________________________________________________________________    N-Alkanoylamino Acid Derivatives of A-30912A Nucleus                           ##STR105##                                                                   Exam-                                                     HPLC                ple Product               Ester     A30912A               Retention           No. R.sup.1 in Formula III                                                                              Example                                                                            Wt. (mg)                                                                           nucleus (mg)                                                                         Product (mg)                                                                         M.sup.+ (cm)                __________________________________________________________________________    61  CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO                                       31   141  250    158    1148(M.sup.+  +                                                                       --)                 62  CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4CO                                                   32   795  250    132    1101(M.sup.+ + 22)                                                                    --                  63  CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10CO                                                  33   462  400    327    1185(M.sup.+  +                                                                       1.23                64                                                                                 ##STR106##           34   515  400    247    1121(M.sup.+ + 23)                                                                    --                  65                                                                                 ##STR107##           35   515  400    302    1120(M.sup.+ + 22)                                                                    --                  66                                                                                 ##STR108##           36   515  400    354    1137(M.sup.+ + 23)                                                                    1.33                67                                                                                 ##STR109##           37   570  400    196    1197(M.sup.+ + 30)                                                                    1.65                68                                                                                 ##STR110##           38   750  400    291    --      1.20                69                                                                                 ##STR111##           39   512  400    182    1135(M.sup.+ + 23)                                                                    --                  70                                                                                 ##STR112##           40   512  400    166    1143(M.sup.+ + 31)                                                                    1.43                71                                                                                 ##STR113##           41   530  400    120    1151(M.sup.+ + 23)                                                                    --                  72                                                                                 ##STR114##           42   497  400    452    1135(M.sup.+ + 23)                                                                    1.32                73                                                                                 ##STR115##           43   535  400    286    1148(M.sup.+ + 24)                                                                    1.40                74                                                                                 ##STR116##           44   540  400    453    1170(M.sup.+ + 25)                                                                    1.40                75                                                                                 ##STR117##           45   492  400    277    --      --                  76  CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10CO                                                   46   493  400    273    1115(M.sup.+ + 23)                                                                    0.95                77                                                                                 ##STR118##           47   360  400    213    980(M.sup.+ +                                                                         1.75                78                                                                                 ##STR119##           48   430  400    218    --      --                  79                                                                                 ##STR120##           49   500  400    162    1093(M.sup.+ + 23)                                                                    0.90                80                                                                                 ##STR121##           50   527  400    234    1149(M.sup.+ + 23)                                                                    2.35                81                                                                                 ##STR122##           51   555  400    350    1177(M.sup.+ + 23)                                                                    3.23                82                                                                                 ##STR123##           52   477  400    176    1099(M.sup.+  +                                                                       0.7                 83                                                                                 ##STR124##           53   533  400    127    1155(M.sup.+ + 23)                                                                    2.3                 84                                                                                 ##STR125##           54   477  400    319    1099(M.sup.+ + 23)                                                                    1.0                 85                                                                                 ##STR126##           55   533  400    214    1155(M.sup.+ + 23)                                                                    3.1                 86                                                                                 ##STR127##           56   477  400    290            1.0                 87                                                                                 ##STR128##           57   533  400    325            3.4                 88                                                                                 ##STR129##           58   512  400    324            1.3                 89                                                                                 ##STR130##           59   540  400    281    1162(M.sup.+ + 23)                                                                    1.8                 90                                                                                 ##STR131##           60   596  400    269    1217(M.sup.+ + 23)                                                                    7.7                 __________________________________________________________________________

EXAMPLE 91

The following procedure illustrates the larger-scale preparation of thecompounds of Formula III. The specific compound prepared by theprocedure given below is the compound of Formula III wherein R¹ isN-(n-dodecanoyl)-p-aminobenzoyl.

A. Preparation of N-(n-Dodecanoyl)-p-aminobenzoic acid

n-dodecanoyl chloride (8.74 g.; 40 mmoles) is added dropwise to asolution of dissolved p-aminobenzoic acid (5.5 g.; 40 mmoles) dissolvedin pyridine (100 ml.). The mixture is stirred for 3 hours and pouredinto water (3 l.). The precipitate which forms is filtered and dried invacuo to give N-(n-dodecanoyl)-p-aminobenzoic acid (11.01 g.).

B. Preparation of the 2,4,5-trichlorophenyl ester ofN-(n-dodecanoyl)-p-aminobenzoic acid

N-(n-Dodecanoyl)-p-aminobenzoic acid (11.01 g.; 34.5 mmole),2,4,5-trichlorophenol (7.5 g.; 38 mmole), and dicyclohexylcarbodiimide(6.94 g.; 34.5 mmole) are dissolved in methylene chloride (250 ml). Themixture is stirred at room temperature for 3.5 hours and then filtered.The filtrate is evaporated in vacuo to give a residue which iscrystallized from acetonitrile/water to afford the 2,4,5-trichlorophenylester of N-(n-dodecanoyl)-p-aminobenzoic acid (12.84 g.).

C. Acylation of A-30912A nucleus

A-30912A nucleus (8.16 g.; 10.2 mmole) and the 2,4,5-trichlorophenylester of N-(n-dodecanoyl)-p-aminobenzoic acid (4.72 g.; 10.2 mmole) aredissolved in dimethylformamide (100 ml.). The solution is stirred atroom temperature for 15 hours. Solvent is removed in vacuo to give aresidue which is washed twice with diethylether. The washes arediscarded. The washed residue is dissolved in methanol (50 ml.) and ispurified by reversed phase HPLC by means of a "Prep LC/System 500" unit(Waters Associates, Inc., Milford, Mass.) using a Prep Pak-500/C₁₈Column (Waters Associates, Inc.) as the stationary phase. The column iseluted isocratically with H₂ O/CH₃ OH/CH₃ CN (25:65:10 v/v) at 500 psi.The fractions are analyzed by TLC using silica gel plates and H₂ O/CH₃OH/CH₃ CN (25:65:10 v/v) as the solvent system. Fractions containing thedesired product are combined and lyophilized to give theN-(n-dodecanoyl)-p-aminobenzoyl derivative of A-30912A nucleus (3.5 g).

EXAMPLE 92

The antifungal activity of the compounds of Formula III can bedemonstrated and elicited in vitro in standard disc-diffusion tests andagar-dilution tests, and in vivo in standard tests in mice which assesseffectiveness against a systemic fungal infection. The results of theantifungal testing of representative compounds of Formula III (Example61-90) are set forth in Tables 4, 5, 6 and 7.

Tables 4 and 5 give the results of the testing in vitro of the compoundsof Examples 61-81 by agar-plate disc-diffusion methods. In Table 4activity is measured by the size (diameter in mm.) of the observed zoneof inhibition of the microorganism produced by the test compound. InTable 5, activity is measured by the minimal inhibitory concentration(MIC) of the substance (μg/disc) required to inhibit growth of the testorganism. Table 6 gives the results of the testing in vitro of theN-(n-dodecanoyl)-p-aminobenzoyl derivative of A30912A nucleus (FormulaIII, R¹ is N-(dodecanoyl)-p-aminobenzoyl) against five strains ofCandida albicans by the agar dilution method. In Table 6 activity ismeasured by the minimal inhibitory concentration (MIC) of the substance(μg/ml) required to inhibit the test organism.

The results of in vivo tests to evaluate the effectiveness of thecompound of Examples 61-81, 86 and 88 against an infection caused byCandida albicans A-26 in mice are given in Table 7, where activity ismeasured by the ED₅₀ value (the dose in mg/kg. required to cure 50% ofthe test animals). Where an ED₅₀ value was not obtained, activity isindicated by the lowest dose at which a significant anti-fungal effectis observed. In this test, groups of male albino mice (specific pathogenfree), weighing 18 to 20 grams, are infected intravenously with Candidaalbicans A-26. The animals are X-irradiated 24 hours prior to infectionat about 50 roentgens per minute for 8 minutes (400 total dose) toreduce immune responses to the infecting organism. At 0, 4, and 24 hourspost infection each group of mice is given graded doses subcutaneouslyof the test compound as a suspension in 33% polyethylene glycol-water.The day of death for each animal is recorded. Student's t teststatistical comparison of the average day of death is made between eachgroup of infected-treated animals at a particular dosage level and 10infected-untreated animals to determine if treatment significantlyextends survival time.

Table 8 gives the results of the testing of compounds for absorptionafter oral administration. In this test, mice are gavaged with a dose of416 mg/kg of the test compound suspended in 33% PEG 400-water. At timeintervals, blood samples are taken from the orbital sinus and areassayed for antibiotic activity as follows: A 7 mm. disc containing 20μl of whole blood is placed on agar seeded with Aspergillusmontevidensis A35137. After 40 hours incubation at 30° C. zones ofinhibition from the blood samples are compared to a standard obtainedfrom the test compound, and the amount of compound in the blood sampleis calculated.

                                      TABLE 4                                     __________________________________________________________________________    Antifungal Activity By the Agar Plate Disc Diffusion Test                     Compound                       Size of Zone of Inhibition (mm).sup.(a)        Example                        Saccharomyces                                                                         Neurospora                                                                           Trichophyton                                                                            Candida               No.  R.sup.1 of Formula III    pastorianus X-52                                                                      Crassa 846                                                                           mentagrophytes                                                                          albicans              __________________________________________________________________________                                                            A-26                  61   CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO                                           18      42*    55*       25                    62   CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4CO                                                       15      27*    60*       25                    63   CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10CO                                                      15      28*    55*       24                                                   18      35     63*       25                                                   15      28     56        24                    64                                                                                  ##STR132##               21 17   33* 35*                                                                              55* 56*   23 19                 65                                                                                  ##STR133##               17      --     --        20                    66                                                                                  ##STR134##               18      --     --        23                    67                                                                                  ##STR135##               19      35*    44*       27                    68                                                                                  ##STR136##               17      30*    60        25                    69                                                                                  ##STR137##               17      30*    --        16                    70                                                                                  ##STR138##               19      25*    45*       26                    71                                                                                  ##STR139##               10      32*    50*       20                    72                                                                                  ##STR140##               20      30*    55*       19                    73                                                                                  ##STR141##               17      29*    24*       60*                   74                                                                                  ##STR142##               13      28*    --        15                    75                                                                                  ##STR143##               14      30*    54*       24                    76   CH.sub.3 (CH.sub.2).sub. 5 CONH(CH.sub.2).sub.10CO                                                      --      20*    35*       10                    77                                                                                  ##STR144##               17      24*    51*       24                    78                                                                                  ##STR145##               20      25     14        45*                   79                                                                                  ##STR146##               17      30*    --        24                    80                                                                                  ##STR147##               17      --     --        22                    81                                                                                  ##STR148##               24      --     --        25                    __________________________________________________________________________     .sup.(a) Compounds were tested as suspension in methanol. The compounds       were tested at a concentration of 1 mg/ml by a dipping 7mm disc into the      suspension and placing it on the agar surface. Incubation: 24-48 hours at     25-37° C.                                                              *Measurable zone of inhibition with regrowth of organism around disc.    

                                      TABLE 5                                     __________________________________________________________________________    Antifungal Activity By the Agar Plate Disc Diffusion Test                     Compound                          MIC (μg/disc)*                           Example No.                                                                          R.sup.1 or Formula III     Candida albicans A-26                                                                     Trychophyton mentagrophytes                                                   #6                              __________________________________________________________________________    61     CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO                                               0.625        <0.039                      62     CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4CO                                                           1.25         0.078                       63     CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10CO                                                          2.5          0.156                                                            1.25         0.156                       64                                                                                    ##STR149##                   0.625 1.25   <0.039 0.678                65                                                                                    ##STR150##                   5.0          <0.039                      66                                                                                    ##STR151##                   1.25         <0.039                      67                                                                                    ##STR152##                   0.625        <0.039                      68                                                                                    ##STR153##                   1.25         0.078                       69                                                                                    ##STR154##                   >20          <0.039                      70                                                                                    ##STR155##                   20           <0.039                      71                                                                                    ##STR156##                   --           --                          72                                                                                    ##STR157##                   2.5          0.078                       73                                                                                    ##STR158##                   1.25         <0.039                      74                                                                                    ##STR159##                   10           0.078                       75                                                                                    ##STR160##                   2.5          0.078                       76     CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10CO                                                           >20;80       0.625                       77                                                                                    ##STR161##                   >20;40       0.312                       78                                                                                    ##STR162##                   >20;160      1.25                        79                                                                                    ##STR163##                   0.625        --                          80                                                                                    ##STR164##                   1.25         <0.039                      81                                                                                    ##STR165##                   0.312        <0.039                      __________________________________________________________________________     *Compounds were suspended in 0.01M sodium borate solution, pH 7.5. The        compounds were tested at 20 μg/disc at top level and at twofold            dilutions until end points were reached. Incubation: 24 hours at              30° C.                                                                 **Measurable zones of inhibition with regrowth of organism around disc.  

TABLE 6

In vitro activity of the N-(n-dodecanoyl)-p-aminobenzoyl (Example 64)and the N-(n-dodecanoyl)-5-amino-n-pentanoyl (Example 62) derivatives ofA-30912A nucleus against 5 strains of Candida albicans by the agardilution assay.

    ______________________________________                                        MIC (μg/ml)                                                                Compound A26    SBH 16   SBH 31 SBH 28  SBH 29                                ______________________________________                                        Ex. 64   0.312  0.625    0.625  0.625   0.625                                 Ex. 62   1.25   2.5      2.5    2.5     2.5                                   ______________________________________                                    

                                      TABLE 7                                     __________________________________________________________________________    Therapeutic Activity Against Candida Albicans A-26 in Mice*                                                       Lowest                                    Compound                            Active                                    Example                       ED.sub.50                                                                           Dose                                      No.  R.sup.1 of Formula III   (mg/kg)**                                                                           (mg/kg)                                   __________________________________________________________________________    61   CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO                                          >40   >40                                       62   CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4CO                                                      22    20                                        63   CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10CO                                                     >40   >40                                       64                                                                                  ##STR166##              15 15 10 ≦5                              65                                                                                  ##STR167##              >40   >40                                       66                                                                                  ##STR168##              >40   40                                        67                                                                                  ##STR169##              14    10                                        68                                                                                  ##STR170##              >40   40                                        69                                                                                  ##STR171##              >40   >40                                       70                                                                                  ##STR172##              >40   >40                                       71                                                                                  ##STR173##              >40   >40                                       72                                                                                  ##STR174##              >40   40                                        73                                                                                  ##STR175##              24    20                                        74                                                                                  ##STR176##              >40   >40                                       75                                                                                  ##STR177##              >40   >40                                       76   CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10CO                                                      >40   >40                                       77                                                                                  ##STR178##              >40   >40                                       78                                                                                  ##STR179##              >40   >40                                       79                                                                                  ##STR180##              26     5                                        80                                                                                  ##STR181##              11    2.5                                       81                                                                                  ##STR182##               7     5                                        86                                                                                  ##STR183##              29    10                                        88                                                                                  ##STR184##              >40   20                                        __________________________________________________________________________     *Dosage Schedule: 40, 20, 15, and 10 mg/kg. Dosages given 0, 4, and 24        hours post injection as suspension of test compound in 30% PEGH.sub.2 O.      Number of mice receiving test compounds at each dosage level: 6 mice per      group. Number of mice in control (untreated) group: 10 mice per group.        **As measured by increase in survival time of treated animals versus          control, calculated by method of Reed V. Mueuch, American J. Hygiene, 493     (1938).                                                                  

                                      TABLE 8                                     __________________________________________________________________________    Blood Levels after Administration in Mice                                     Compound                                                                      Example                                                                       No.  R.sup.1 in Formula III   Blood Levels* (μg/ml)                        __________________________________________________________________________    61   CH.sub.3 (CH.sub.2).sub.10 CONHCH(CH.sub.2 C.sub.6 H.sub.5)CO                                          0                                               62   CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.4CO                                                      0                                               63   CH.sub.3 (CH.sub.2).sub.10 CONH(CH.sub.2).sub.10                                                       0.40(0)                                         64                                                                                  ##STR185##              0.83                                            65                                                                                  ##STR186##              0                                               66                                                                                  ##STR187##              0                                               67                                                                                  ##STR188##              0.53                                            68                                                                                  ##STR189##              0.34                                            69                                                                                  ##STR190##              0                                               70                                                                                  ##STR191##              0                                               71                                                                                  ##STR192##              --                                              72                                                                                  ##STR193##              0.34                                            73                                                                                  ##STR194##              1.31                                            74                                                                                  ##STR195##              0.64                                            75                                                                                  ##STR196##              0                                               76   CH.sub.3 (CH.sub.2).sub.5 CONH(CH.sub.2).sub.10CO                                                      0                                               77                                                                                  ##STR197##              0                                               78                                                                                  ##STR198##              --                                              79                                                                                  ##STR199##              --                                              80                                                                                  ##STR200##              6.5                                             81                                                                                  ##STR201##              36                                              __________________________________________________________________________     *Four hours after administration of test compound at dose of 416 mg/kg by     gavage as suspension of compound in 33% PEG 400H.sub.2 O). Compound           determined by bioassay vs. Aspergillus montevidensis A35137.             

EXAMPLE 93 Preparation of A-30912A Nucleus A. Fermentation ofActinoplanes utahensis NRRL 12052

A stock culture of Actinoplanes utahensis NRRL 12052 is prepared andmaintained on an agar slant. The medium used to prepare the slant isselected from one of the following:

    ______________________________________                                        MEDIUM A                                                                      Ingredient             Amount                                                 ______________________________________                                        Baby oatmeal           60.0 g                                                 Yeast                   2.5 g                                                 K.sub.2 HPO.sub.4       1.0 g                                                 Czapek's mineral stock*                                                                               5.0 ml                                                Agar                   25.0 g                                                 Deionized water        q.s. to 1 liter                                        ______________________________________                                        pH before autoclaving is about 5.9; adjust to pH 7.2 by                       addition of NaOH; after autoclaving, pH is about 6.7.                         *Czapek's mineral stock has the following composition:                        Ingredient             Amount                                                 ______________________________________                                        FeSO.sub.4 . 7H.sub.2 O (dissolved in                                         2 ml conc HCl)          2 g                                                   KCl                    100 g                                                  MgSO.sub.4 . 7H.sub.2 O                                                                              100 g                                                  Deionized water        q.s. to 1 liter                                        ______________________________________                                    

    ______________________________________                                        MEDIUM B                                                                      Ingredient               Amount                                               ______________________________________                                        Potato dextrin           5.0    g                                             Yeast extract            0.5    g                                             Enzymatic hydrolysate of casein*                                                                       3.0    g                                             Beef extract             0.5    g                                             Dextrose                 12.5   g                                             Corn starch              5.0    g                                             Meat peptone             5.0    g                                             Blackstrap molasses      2.5    g                                             MgSO.sub.4 . 7H.sub.2 O  0.25   g                                             CaCO.sub.3               1.0    g                                             Czapek's mineral stock   2.0    ml                                            Agar                     20.0   g                                             Deionized water          q.s. to 1 liter                                      ______________________________________                                         *N--Z--Amine A, Humko Sheffield Chemical, Lyndhurst, N.J.                

The slant is inoculated with Actinoplanes utahensis NRRL 12052 and theinoculated slant is incubated at 30° C. for about 8 to 10 days. About1/2 of the slant growth is used to inoculate 50 ml of a vegetativemedium having the following composition:

    ______________________________________                                        Ingredient             Amount                                                 ______________________________________                                        Baby oatmeal           20.0     g                                             Sucrose                20.0     g                                             Yeast                  2.5      g                                             Distiller's Dried Grain*                                                                             5.0      g                                             K.sub.2 HPO.sub.4      1.0      g                                             Czapek's mineral stock 5.0      ml                                            Deionized water        q.s. to 1 liter                                        adjust to pH 7.4 with NaOH; after autoclaving, pH is about                    ______________________________________                                        6.8                                                                            *National Distillers Products Co., 99 Park Ave., New York, N.Y.          

The inoculated vegetative medium is incubated in a 250-ml wide-mouthErlenmeyer flask at 30° C. for about 72 hours on a shaker rotatingthrough an arc two inches in diameter at 250 RPM.

This incubated vegetative medium may be used directly to inoculate asecond-stage vegetative medium. Alternatively and preferably, it can bestored for later use by maintaining the culture in the vapor phase ofliquid nitrogen. The culture is prepared for such storage in multiplesmall vials as follows: In each vial is placed 2 ml of incubatedvegetative medium and 2 ml of a glycerol-lactose solution [see W. A.Dailey and C. E. Higgens, "Preservation and Storage of Microorganisms inthe Gas Phase of Liquid Nitrogen, Cryobiol 10, 364-367 (1973) fordetails]. The prepared suspensions are stored in the vapor phase ofliquid nitrogen.

A stored suspension (1 ml) thus prepared is used to inoculate 50 ml of afirst-stage vegetative medium (having the composition earlierdescribed). The inoculated first-stage vegetative medium is incubated asabove-described.

In order to provide a larger volume of inoculum, 10 ml of the incubatedfirst-stage vegetative medium is used to inoculate 400 ml of asecond-stage vegetative medium having the same composition as thefirst-stage vegetative medium. The second-stage medium is incubated in atwo-liter wide-mouth Erlenmeyer flask at 30° C. for about 48 hours on ashaker rotating through an arc two inches in diameter at 250 RPM.

Incubated second-stage vegetative medium (800 ml), prepared asabove-described, is used to inoculate 100 liters of sterile productionmedium selected from one of the following:

    ______________________________________                                        MEDIUM I                                                                      Ingredient           Amount (g/L)                                             ______________________________________                                        Peanut meal          10.0                                                     Soluble meat peptone 5.0                                                      Sucrose              20.0                                                     KH.sub.2 PO.sub.4    0.5                                                      K.sub.2 HPO.sub.4    1.2                                                      MgSO.sub.4 . 7H.sub.2 O                                                                            0.25                                                     Tap water            q.s. to 1 liter                                          ______________________________________                                    

The pH of the medium is about 6.9 after sterilization by autoclaving at121° C. for 45 minutes at about 16-18 psi.

    ______________________________________                                        MEDIUM II                                                                     Ingredient          Amount (g/L)                                              ______________________________________                                        Sucrose             30.0                                                      Peptone             5.0                                                       K.sub.2 HPO.sub.4   1.0                                                       KCl                 0.5                                                       MgSO.sub.4 . 7H.sub.2 O                                                                           0.5                                                       FeSO.sub.4 . 7H.sub.2 O                                                                           0.002                                                     Deionized water     q.s. to 1 liter                                           Adjust to pH 7.0 with HCl; after autoclaving, pH is                           about 7.0                                                                     ______________________________________                                    

    ______________________________________                                        MEDIUM II                                                                     Ingredient          Amount (g/L)                                              ______________________________________                                        Glucose             20.0                                                      NH.sub.4 Cl         3.0                                                       Na.sub.2 SO.sub.4   2.0                                                       ZnCl.sub.2          0.019                                                     MgCl.sub.2 . 6H.sub.2 O                                                                           0.304                                                     FeCl.sub.3 . 6H.sub.2 O                                                                           0.062                                                     MnCl.sub.2 . 4H.sub.2 O                                                                           0.035                                                     CuCl.sub.2 . 2H.sub.2 O                                                                           0.005                                                     CaCO.sub.3          6.0                                                       KH.sub.2 PO.sub.4 * 0.67                                                      Tap water           q.s. to 1 liter                                           ______________________________________                                         *Sterilized separately and added aseptically                             

Final pH about 6.6.

The inoculated production medium is allowed to ferment in a 165-literfermentation tank at a temperature of about 30° C. for about 42 hours.The fermentation medium is stirred with conventional agitators at about200 RPM and aerated with sterile air to maintain the dissolved oxygenlevel above 30% of air saturation at atmospheric pressure.

B. Deacylation of Antibiotic A-30912 Factor A

A fermentation of A. utahensis is carried out as described in Sect. A,using slant medium A and production medium I and incubating theproduction medium for about 42 hours. A-30912 factor A (340 g. of crudesubstrate which contained about 19.7 g. of A-30912 factor A, dissolvedin 1.5 L ethanol) is added to the fermentation medium.

Deacylation of A-30912 factor A is monitored by assay against Candidaalbicans. The fermentation is allowed to continue until deacylation iscomplete as indicated by disappearance of activity vs. C. albicans.

c. Isolation of A-30912A Nucleus

Whole fermentation broth (100 liters), obtained as described in Sect. Band containing nucleus from about 20 g of A-30912 factor A, is filtered.The mycelial cake is discarded. The clear filtrate thus obtained (about93 liters) is passed through a column containing 4.5 liters of HP-20resin (DIAION High Porous Polymer, HP-Series, Mitsubishi ChemicalIndustries Limited, Tokyo, Japan) at a rate of 200 ml/minute. Theeffluent thus obtained is discarded. The column is then washed with upto eight column volumes of deionized water at pH 6.5-7.5 to removeresidual filtered broth. This wash water is discarded. The column isthen eluted with a water:methanol (7:3) solution (85 liters) at a rateof 200-300 ml/minute.

Elution is monitored using the following procedure: Two aliquots aretaken from each eluted fraction. One of the aliquots is concentrated toa small volume and is treated with an acid chloride such as myristoylchloride. This product and the other (untreated) aliquot are assayed foractivity against Candida albicans. If the untreated aliquot does nothave activity and the acylated aliquot does have activity, the fractioncontains A-30912A nucleus. The eluate containing the A-30912A nucleus isconcentrated under vacuum to a small volume and lyophilized to giveapproximately 97 grams of crude nucleus.

D. Purification of A-30912A Nucleus by Reversed-Phase LiquidChromatography

Crude A-30912A nucleus (25 grams), obtained as described in Section C,is dissolved in 300 ml of water:acetonitrile:acetic acid:pyridine(96:2:1:1). This solution is chromatographed on a 4-literstainless-steel column (8 cm×80 cm) filled with Lichroprep RP-18,particle size 25-40 microns (MC/B Manufacturing Chemists, Inc. E/M,Cincinnati, OH). The column is part of a Chromatospac Prep 100 unit(Jobin Yvon, 16-18 Rue du Canal 91160 Longjumeau, France). The column isoperated at a pressure of 90-100 psi, giving a flow rate of about 60ml/minute, using the same solvent. Separation is monitored at 280 nmusing a UV monitor (ISCO Absorption Monitor Model UA-5, InstrumentSpecialist Co., 4700 Superior Ave., Lincoln, Nebr. 68504) with anoptical unit (ISCO Type 6). Fractions having a volume of about 500 mlare collected each minute.

On the basis of absorption at 280 nm, fractions containing A-30912Anucleus are combined, evaporated under vacuum and lyophilized to give2.6 grams of nucleus. The amount of solvent required to complete thischromatographic separation process varies from 7-8 liters.

E. Characteristics of A30912A nucleus

(a) Empirical formula: C₃₄ H₅₁ N₇ O₁₅.

(b) Molecular weight: 797.83

(c) Soluble in water, dimethylformamide, dimethylsulfoxide, andmethanol; insoluble in chloroform, toluene, and diethylether.

(d) Infrared absorption spectrum (KBr disc.)

Shows absorption maxima at:

3340 broad (OH, H-bonded); 2970, 2930, and 2890 (CH stretch, aliphaticCH₃, CH₂, CH groups) 1660 and 1625 (several carbonyls C═O); 1510-1550;1430-1450 (CH wag); 1310-1340; 1230-1260; 1080; 835, 650 broad, and 550broad cm⁻¹.

(e) Electrometric titration in 66% aqueous dimethylformamide indicatesthe presence of a titratable group with a pK_(a) value of about 7.35(initial pH 7.32).

(f) HPLC retention time (K'):11.52 min. under following conditions.

Column: 4×300 mm

Packing: silica gel/C₁₈

Solvent: ammonium acetate:acetonitrile:

water (1:2:97)

Flow Rate: 3 ml/min

Pressure: 2500 psi

Detector: variable wavelength UV at 230 nm

Sensitivity: 0-0.4 A.U.F.S.

EXAMPLE 94

A-30912A nucleus is prepared and purified by the method of Example 93except that tetrahydro-A-30912A is used as the substrate.

EXAMPLE 95

A-30912A nucleus is prepared and purified by the method of Example 93except that aculeacin A is used as the substrate.

EXAMPLE 96 Preparation of the A-42355 Antibiotic Complex A. Shake-FlaskFermentation

A culture of Aspergillus nidulans var. roseus NRRL 11440 is prepared andmaintained on an agar slant prepared with medium having the followingcomposition:

    ______________________________________                                        Ingredient           Amount                                                   ______________________________________                                        Glucose               5 g                                                     Yeast extract         2 g                                                     CaCO.sub.3            3 g                                                     Vegetable juice*     200 ml                                                   Agar**               20 g                                                     Deionized water      q.s. to 1 liter                                          (initial pH 6.1)                                                              ______________________________________                                         *V-8 Juice, Campbell Soup Co., Camden, N.J.                                   **Meer Corp.                                                             

The slant is inoculated with Aspergillus nidulans var. roseus NRRL11440, and the inoculated slant is incubated at 25° C. for about sevendays. The mature slant culture is covered with water and scraped with asterile loop to loosen the spores. The resulting suspension is furthersuspended in 10 ml of sterile deionized water.

One ml of the suspended slant growth is used to inoculate 55 ml ofvegetative medium in a 250-ml flask. The vegetative medium has thefollowing composition:

    ______________________________________                                        Ingredient             Amount                                                 ______________________________________                                        Sucrose                25 g                                                   Blackstrap molasses    36 g                                                   Corn-steep liquor       6 g                                                   Malt extract           10 g                                                   K.sub.2 HPO.sub.4       2 g                                                   Enzymatic hydrolysate                                                         of casein*             10 g                                                   Tap water              1100 ml                                                (initial pH 6.5-6.7)                                                          ______________________________________                                         *N-Z-Case, Humko Sheffield Chemical, Lyndhurst, N.J.                     

The inoculated vegetative medium is incubated at 25° C. for 48 hours at250 rpm on a rotary-type shaker. After 24 hours, the medium ishomogenized for one minute at low speed in a blender (Waring type) andthen returned to incubation for the remaining 24 hours. Alternatively,the inoculated vegetative medium can be incubated for 48 hours and thenhomogenized for 15 seconds at low speed.

This incubated vegetative medium may be used to inoculate shake-flaskfermentation culture medium or to inoculate a second-stage vegetativemedium. Alternatively, it can be stored for later use by maintaining theculture in the vapor phase of liquid nitrogen. The culture is preparedfor such storage in multiple small vials as follows:

The vegetative cultures are mixed volume/volume with a suspendingsolution having the following composition:

    ______________________________________                                        Ingredient           Amount                                                   ______________________________________                                        Glycerol             20 ml                                                    Lactose              10 g                                                     Deionized water      q.s. to 100 ml                                           ______________________________________                                    

The prepared suspensions are distributed in small sterile screw-captubes (4 ml per tube). These tubes are stored in the vapor phase ofliquid nitrogen.

A stored suspension thus prepared can be used to inoculate either agarslants or liquid seed media. Slants are incubated at 25° C. in the lightfor 7 days.

B. Tank Fermentation

In order to provide a larger volume of inoculum, 10 ml of incubatedfirst-stage vegetative culture is used to inoculate 400 ml of asecond-stage vegetative growth medium having the same composition asthat of the vegetative medium. The second-stage medium is incubated in atwo-liter wide-mouth Erlenmeyer flask at 25° C. for 24 hours on a shakerrotating through an arc two inches in diameter at 250 rpm.

Incubated second-stage medium (800 ml), prepared as above described, isused to inoculate 100 liters of sterile production medium selected fromone of the following:

    ______________________________________                                        MEDIUM IV                                                                     Ingredient          Amount                                                    ______________________________________                                        ZnSO.sub.4 . 7H.sub.2 O                                                                           0.00455  g/L                                              Soluble meat peptone*                                                                             30.5     g/L                                              Soybean meal        15.5     g/L                                              Tapioca dextrin**   2.0      g/L                                              Blackstrap molasses 10.5     g/L                                              Enzymatic hydrolysate                                                         of casein***        8.5      g/L                                              Na.sub.2 HPO.sub.4  4.5      g/L                                              MgSO.sub.4 . 7H.sub.2 O                                                                           5.5      g/L                                              FeSO.sub.4 . 7H.sub.2 O                                                                           0.1      g/L                                              Cottonseed oil      40.0     ml                                               (Antifoam)****      1.0      ml                                               Tap water           1000.0   ml                                               (initial pH 6.8-7.0)                                                          ______________________________________                                         *O.M. Peptone, Amber Laboratories, Juneau, Wisc.                              **Stadex 11, A.E. Staley Co., Decatur, Ill.                                   ***N-Z-Amine A, Humko Sheffield Chemical, Lyndhurst, N.J.                     **** P2000, Dow Corning, Midland, Michigan                               

    ______________________________________                                        MEDIUM V                                                                      Ingredient            Amount                                                  ______________________________________                                        Glucose               2.5%                                                    Starch                1.0%                                                    Soluble meat peptone* 1.0%                                                    Blackstrap molasses   1.0%                                                    CaCO.sub.3            0.2%                                                    MgSO.sub.4 . 7H.sub.2 O                                                                             0.05%                                                   Enzymatic hydrolysate of                                                      casein**              0.4%                                                    (Antifoam)***         0.02%                                                   Tap water             q.s. to volume                                          ______________________________________                                         *O.M. Peptone                                                                 **N-Z-Amine A                                                                 ***Antifoam "A", Dow Corning                                             

The inoculated production medium is allowed to ferment in a 165-literfermentation tank at a temperature of 25° C. for about 7 days. Thefermentation medium is aerated with sterile air, maintaining thedissolved oxygen level above approximately 50 percent of air saturation.

C. Third-Stage Vegetative Medium

Whenever the fermentation is carried out in tanks larger than those usedfor 100-liter fermentation, it is recommended that a third-stagevegetative culture be used to seed the larger tank. A preferredthird-stage vegetative medium has the following composition:

    ______________________________________                                        Ingredient           Amount                                                   ______________________________________                                        Sucrose              25 g                                                     Blackstrap molasses  25 g                                                     Corn-steep liquor     6 g                                                     Enzymatic hydrolysate                                                         of casein*           10 g                                                     Malt extract         10 g                                                     K.sub.2 HPO.sub.4     2 g                                                     Tap water            1000 ml                                                  (initial pH 6.1)                                                              ______________________________________                                         *N-Z-Case                                                                

EXAMPLE 97 Separation of the A-42355 Antibiotic Complex

Whole fermentation broth (4127 liters), obtained by the method describedin Example 96 using production medium V, is stirred thoroughly withmethanol (4280 liters) for one hour and then is filtered, using a filteraid (Hyflo Super-cel, a diatomaceous earth, Johns-Manville ProductsCorp.). The pH of the filtrate is adjusted to pH 4.0 by the addition of5 N HCl. The acidified filtrate is extracted twice with equal volumes ofchloroform. The chloroform extracts are combined and concentrated undervacuum to a volume of about 20 liters. This concentrate is added toabout 200 l liters of diethyl ether to precipitate the A-42355 complex.The precipitate is separated by filtration to give 2775 g of the A-42355complex as a gray-white powder.

EXAMPLE 98 Isolation of A-30912 Factor A

The co-pending application of Karl H. Michel entitled RECOVERY PROCESSFOR A-30912 ANTIBIOTICS, Ser. No. 103,014, filed Dec. 13, 1979,describes the reversed-phase high performance, low pressure liquidchromatography (HPLPLC) using silica gel/C₁₈ adsorbent as a preferredmethod for the final purification of A-30912 factor A.

A-42355 antibiotic complex (1 g), prepared as described in Example 97,is dissolved in 7 ml of methanol:water:acetonitrile (7:2:1). Thissolution is filtered and introduced onto a 3.7-cm I.D.×35-cm glasscolumn [Michel-Miller High Performance Low Pressure (HPLPLC)Chromatography Column, Ace Glass Incorporated, Vineland, NJ 08360]packed with LP-1/C₁₈ silica gel reversed-phase resin (10-20 microns),prepared as described in Example 99, through a loop with the aid of avalve system. The column is packed in methanol:water:acetonitrile(7:2:1) by the slurry-packing procedure described in Example 100. AnF.M.I. pump with valveless piston design (maximum flow 19.5 ml/minute)is used to move the solvent through the column at a flow rate of 9ml/minute at ca. 100 psi, collecting fractions every minute. Elution ofthe antibiotic is monitored at 280 nm by using a UV monitor (ISCO ModelUA-5, Instrument Specialist Co., 4700 Superior Ave., Lincoln, Nebr.68504) with an optical unit (ISCO Type 6).

The individual A-30912 factors can be identified by the use ofthin-layer chromatography (TLC). The R_(f) values of A-30912 factorsA-G, using silica gel (Merck, Darmstadt) TLC, a benzene:methanol (7:3)solvent system, and Candida albicans bioautography are given in Table 9.

                  TABLE 9                                                         ______________________________________                                        A-30912 Factor    R.sub.f Value                                               ______________________________________                                        A                 0.35                                                        B                 0.45                                                        C                 0.54                                                        D                 0.59                                                        E                 0.27                                                        F                 0.18                                                        G                 0.13                                                        ______________________________________                                    

The approximate R_(f) values of A-30912 factors A, B, C, D, and H indifferent solvent systems, using silica gel TLC (Merck-Darmstadt silicagel #60 plates, 20×20 cm) and Candida albicans bioautography, are givenin Table 10.

                  TABLE 10                                                        ______________________________________                                                  R.sub.f Values - Solvent Systems                                    A-30912 Factor                                                                            a        b        c      d                                        ______________________________________                                        Factor A    0.28     0.14     0.28   0.43                                     Factor B    0.39     0.21     0.42   0.47                                     Factor C    0.46     0.31     0.51   0.58                                     Factor D    0.50     0.38     0.57   0.61                                     Factor H    0.42     0.27     0.36   0.53                                     ______________________________________                                         Solvent Systems                                                               a: ethyl acetate:methanol(3:2)                                                b: ethyl acetate:methanol (7:3)                                               c: acetonitrile:water (95:5)                                                  d: ethyl acetate:ethanol:acetic acid (40:60:0.25)                        

A-30912 factors A, B, D and H can also be indentified by analyticalHPLPLC using the following conditions:

    ______________________________________                                        Column:         glass, 0.8 × 15.0 cm                                    Packing:        Nucleosil® 10-C.sub.18 (Machery-                                          Nagel and Company); packed                                                    using slurry-packing pro-                                                     cedure of Example 73                                          Solvent:        methanol:water:aceto-                                                         nitrile (7:2:1)                                               Sample Volume:  8 mcl                                                         Sample Size:    8 mcg                                                         Column Temperature:                                                                           ambient                                                       Flow Rate:      1.8 ml/min                                                    Pressure:       ca. 200 psi                                                   Detector:       UV at 222 nm (ISCO Model                                                      1800 Variable Wavelength                                                      UV-Visible Absorbance                                                         Monitor)                                                      Pump:           LDC Duplex Minipump                                           Injection:      loop injection                                                ______________________________________                                    

The approximate retention times for A-30912 factors A, B, D, and H underthese conditions are summarized in Table 11.

                  TABLE 11                                                        ______________________________________                                                          Retention Time                                              A-30912 Factor    (seconds)                                                   ______________________________________                                        A                 792                                                         B                 870                                                         H                 990                                                         D                 1,140                                                       ______________________________________                                    

EXAMPLE 99 Preparation of Silica Gel/C₁₈ Reversed Phase Resin Step 1:Hydrolysis

LP-1 silica gel (1000 g from Quantum Corp., now Whatman) is added to amixture of concentrated sulfuric acid (1650 ml) and concentrated nitricacid (1650 ml) in a 5-L round-bottom flask and shaken for propersuspension. The mixture is heated on a steam bath overnight (16 hours)with a water-jacketed condenser attached to the flask.

The mixture is cooled in an ice bath and carefully filtered using asintered-glass funnel. The silica gel is washed with deionized wateruntil the pH is neutral. The silica gel is then washed with acetone (4L) and dried under vacuum at 100° C. for 2 days.

Step 2: First Silylation

The dry silica gel from Step 1 is transferred to a round-bottom flaskand suspended in toluene (3.5 L). The flask is heated on a steam bathfor 2 hours to azeotrope off some residual water.Octadecyltrichlorosilane (321 ml, Aldrich Chemical Company) is added,and the reaction mixture is refluxed overnight (16 hours) with slowmechanical stirring at about 60° C. Care is taken so that the stirrerdoes not reach near the bottom of the flask. This is to prevent grindingthe silica gel particles.

The mixture is allowed to cool. The silanized silica gel is collected,washed with toluene (3 L) and acetone (3 L), and then air-driedovernight (16-20 hours). The dried silica gel is suspended in 3.5 L ofacetonitrile:water (1:1) in a 5-L flask, stirred carefully at roomtemperature for 2 hours, filtered, washed with acetone (3 L) andair-dried overnight.

Step 3: Second Silylation

The procedure from the first silylation is repeated using 200 ml ofoctadecyltrichlorosilane. The suspension is refluxed at 60° C. for 2hours while stirring carefully. The final product is recovered byfiltration, washed with toluene (3 L) and methanol (6 L), and then driedunder vacuum at 50° C. overnight (16-20 hours).

EXAMPLE 100 Slurry Packing Procedure for Michel-Miller Columns GeneralInformation

This procedure is employed for packing reversed phase silica gel C₁₈resins, such as that described in Example 99.

Generally, a pressure of less than 200 psi and flow rates between 5-40ml/minute are required for this slurry packing technique; this isdependent on column volume and size. Packing pressure should exceed thepressure used during actual separation by 30-50 psi; this will assure nofurther compression of the adsorbent during separation runs.

A sudden decrease in pressure may cause cracks or channels to form inthe packing material, which would greatly reduce column efficiency.Therefore, it is important to let the pressure drop slowly to zerowhenever the pump is turned off.

The approximate volume of columns (Ace Glass Cat. No., unpacked) are No.5795-04, 12 ml; No. 5795-10, 110 ml; No. 5795-16, 300 ml; No. 5795-24,635 ml; and No. 5796-34, 34 ml.

The time required to pack a glass column will vary from minutes toseveral hours depending on column size and the experience of thescientist.

EXAMPLE

1. Connect glass column to a reservoir column via coupling (volume ofreservoir column should be twice that of the column). Place both columnsin vertical positions (reservoir column above).

2. Weigh out packing material (ca. 100 g for 200 ml column).

3. Add ca. five volumes of solvent to packing material; use a mixture of70-80% methanol and 20-30% water.

4. Shake well until all particles are wetted, let stand overnight orlonger to assure complete soaking of particles by solvent. Decantsupernatant liquid.

5. Slurry the resin with sufficient solvent to fill reservoir column.Pour swiftly into reservoir. The column must be pre-filled with the samesolvent and the reservoir column should be partly filled with solventbefore slurry is poured. The use of larger slurry volumes may alsoprovide good results; however, this will require (a) larger reservoir or(b) multiple reservoir fillings during the packing procedure.

6. Close reservoir with the Teflon plug beneath the column (see FIG. 1of U.S. Pat. No. 4,131,547, plug No. 3); connect to pump; andimmediately start pumping solvent through system at maximum flow rate ifAce Cat. No. 13265-25 Pump or similar solvent-delivery system is used(ca. 20 ml/minute).

7. Continue until column is completely filled with adsorbent. Pressureshould not exceed maximum tolerance of column during this operation (ca.200 psi for large columns and 300 psi for analytical columns). In mostcases, pressures less than 200 psi will be sufficient.

8. Should pressure exceed maximum values, reduce flow-rate; pressurewill drop.

9. After column has been filled with adsorbent, turn off pump; letpressure drop to zero; disconnect reservoir; replace reservoir with apre-column; fill pre-column with solvent and small amount of adsorbent;and pump at maximum pressure until column is completely packed. Foradditional information, see general procedure. Always allow pressure todecrease slowly after turning off pump--this will prevent formation ofany cracks or channels in the packing material.

10. Relieve pressure and disconnect precolumn carefully. With smallspatula remove a few mm (2-4) of packing from top of column; place 1 or2 filter(s) in top of column; gently depress to top of packing material,and place Teflon plug on top of column until seal is confirmed. Connectcolumn to pump, put pressure on (usually less than 200 psi) and observethrough glass wall on top of column if resin is packing any further. Ifpacking material should continue to settle (this may be the case withlarger columns), some dead space or channelling will appear and step 9should be repeated.

EXAMPLE 101 Preparation of Tetrahydro-A-30912A

A-30912 factor A is dissolved in ethanol. PtO₃ in absolute ethanol isreduced to form Pt, which in turn is used to reduce the A-30912 factor Acatalytically, using hydrogenation under positive pressure until thereaction is complete (about 2-3 hours). The reaction mixture is filteredand concentrated under vacuum. The residue is dissolved in a smallamount of tert-butanol and lyophilized to give tetrahydro-A-30912A.

What is claimed is:
 1. A compound of the formula: ##STR202## wherein R¹is an N-alkanoyl amino acyl group of the formula ##STR203## wherein: Wis a divalent aminoacyl radical of the formula: ##STR204## wherein A isC₁ -C₁₀ alkylene or C₅ -C₆ cycloalkylene; ##STR205## wherein R³ ishydroxymethyl, hydroxyethyl, mercaptomethyl, mercaptoethyl,methylthioethyl, 2-thienyl, 3-indole-methyl, phenyl, benzyl, orsubstituted phenyl or substituted benzyl in which the benzene ringthereof is substituted with chloro, bromo, iodo, nitro, C₁ -C₃ alkyl,hydroxy, C₁ -C₃ alkylthio, carbamyl, or C₁ -C₃ alkylcarbamyl; ##STR206##wherein X is hydrogen, chloro, bromo, iodo, nitro, C₁ -C₃ alkyl,hydroxy, C₁ -C₃ alkoxy, mercapto, C₁ -C₃ alkylthio, carbamoyl, or C₁ -C₃alkylcarbamyl; ##STR207## wherein X¹ is chloro, bromo, or iodo;##STR208## wherein B is a divalent radical of the formula; --(CH₂)_(n)--, wherein n is an integer from 1 to 3; --CH═CH--; --CH═CH--CH₂ --; or##STR209## and R² is C₁ -C₁₇ alkyl or C₂ -C₁₇ alkenyl.
 2. A compound asdefined in claim 1 wherein R¹ is ##STR210## wherein A is C₁ -C₁₀alkylene and R² is straight chain C₁ -C₁₇ alkyl.
 3. The compound asdefined in claim 2 wherein R¹ is N-(n-dodecanoyl)-5-amino-n-pentanoyl.4. The compound as defined in claim 2 wherein R¹ isN-(n-dodecanoyl)-11-amino-n-hendecanoyl.
 5. The compound as defined inclaim 2 wherein R¹ is N-(n-heptanoyl)-11-amino-n-hendecanoyl.
 6. Acompound as defined in claim 1 wherein R¹ is ##STR211## wherein X ishydrogen and R₂ is straight chain C₁ -C₁₇ alkyl.
 7. The compound asdefined in claim 6 wherein R¹ is N-(n-dodecanoyl)-p-aminobenzoyl.
 8. Thecompound as defined in claim 6 wherein R¹ isN-(n-dodecanoyl)-m-aminobenzoyl.
 9. The compound as defined in claim 6wherein R¹ is N-(acetyl)-p-aminobenzoyl.
 10. The compound as defined inclaim 6 wherein R¹ is N-(n-heptanoyl)-p-aminobenzoyl.
 11. The compoundas defined in claim 6 wherein R¹ is N-(n-decanoyl)-p-aminobenzoyl. 12.The compound as defined in claim 6 wherein R¹ isN-(n-tetradecanoyl)-p-aminobenzoyl.
 13. The compound as defined in claim6 wherein R¹ is N-(n-hexadecanoyl)-p-aminobenzoyl.
 14. A compound asdefined in claim 1 wherein R¹ is ##STR212## wherein X is chloro, bromo,iodo, nitro, C₁ -C₃ alkyl, hydroxy, C₁ -C₃ alkoxy, mercapto, C₁ -C₃alkylthio, carbamyl, or C₁ -C₃ alkylcarbamyl, and R² is straight chainC₁ -C₁₇ alkyl.
 15. The compound as defined in claim 14 wherein R¹ isN-(n-dodecanoyl)-4-amino-2-hydroxybenzoyl.
 16. The compound as definedin claim 14 wherein R¹ is N-(n-dodecanoyl)-3-amino-4-methylbenzoyl. 17.The compound as defined in claim 14 wherein R¹ isN-(n-dodecanoyl)-4-amino-3-methylbenzoyl.
 18. The compound as defined inclaim 14 wherein R¹ is N-(n-dodecanoyl)-3-amino-4-methoxybenzoyl. 19.The compound as defined in claim 1 wherein R¹ isN-(n-dodecanoyl)-3-amino-2,5-dichlorobenzoyl.
 20. The compound asdefined in claim 1 wherein R¹ isN-(n-dodecanoyl)-4-amino-3,5-diodobenzoyl.
 21. The compound as definedin claim 1 wherein R¹ is N-(n-dodecanoyl)-p-aminophenylacetyl.
 22. Thecompound as defined in claim 1 wherein R¹ isN-(n-dodecanoyl)-p-aminocinnamoyl.
 23. The compound as defined in claim1 wherein R¹ is N-(n-dodecanoyl)-p-aminohippuryl.
 24. The compound asdefined in claim 1 wherein R¹ is N-(n-dodecanoyl)-2-aminonicotinyl. 25.The compound as defined in claim 1 wherein R¹ isN-(n-dodecanoyl)phenylalanyl.